We propose to study biotin holoenzyme synthesis in Saccharomyces cervisiae and E. coli. Our primary objective is to elucidate the recognition mechanism which allows a single holoenzyme synthetase to incorporate biotin into many different biotin-requiring enzymes. We will use two experiment approaches: 1) the primary amino acid sequence will be determined for biotin containing peptides from three biotin-requiring enzymes of Saccharomyces. 2) the biotin carboxyl carrier subunit of E.coli acetyl CoA carboxylase will be prepared in the apoenzyme form. Its ability to serve as apoenzyme substrate for the holoenzyme synthetase will be measured in the presence and absence of the other subunits and after limited and extensive proteolytic digestion.